ABSTRACT In neural circuits, adaptive responses to changes in transmitted signals are conveyed by subtle modulations in the strength of synaptic connections. This short-term synaptic plasticity involves adjustments of the neurotransmitter release probability of synaptic vesicles (SVs) positioned in presynaptic areas called active zones (AZ). A key function of the AZ molecular machinery is to precisely position diffusing membrane voltage-gated calcium channels (VGCCs) in registry with primed SVs, so as to establish the local Ca2+ concentration gradients that ultimately initiate SV fusion and neurotransmitter release. Previous studies have shown that active zone cytomatrix (CAZ) proteins are determinants of the spatial coupling between VGCCs and SVs and that the mobility of VGCCs can tune the SV release probability by setting local channel densities and Ca2+ concentrations. While this suggests that modulation of VGCC dynamics by CAZ proteins could underlie presynaptic plasticity, a fundamental, yet still unanswered question in synaptic biology is how CAZ proteins regulate the mobility of VGCCs and precisely positioned them within AZ a few hundreds of nanometer in size, in the first place. An important challenge in addressing this question has been the absence of methods that allow direct visualization and quantification of VGCC dynamics at the nanometer scale within AZ of intact synapses in live animals. Using CRISPR genetics and complementation activated light microscopy (CALM), an in vivo single molecule (SM) imaging technique that we introduced recently, we have started to define how the molecular machinery of AZ modulates the nanoscale mobility of VGCCs using the nematode Caenorhabditis elegans (C.elegans) as a live animal model. Our preliminary data demonstrate that neuronal VGCCs have heterogeneous diffusive behaviors in vivo, and that their nanoscale mobility is effectively controlled by key CAZ proteins. Here, we built on this preliminary work to further dissect the molecular mechanisms by which different CAZ proteins specifically regulate the presynaptic membrane dynamics of VGCCs in order to guaranty precise neurotransmission. Specifically, we will determine how VGCC dynamics are regulated by (i) the CAZ protein RIM/UNC-10, (ii) coupling to SVs and (iii) coupling to other CAZ regulators (Aim 1), how the priming levels of SVs at AZ influence the mobility of VGCCs (Aim 2), and how the presynaptic dense projection centered in the AZ modulates the nanoconfinement zones of diffusing VGCCs. (Aim 3). Together, the proposed studies will advance our fundamental understanding of the molecular organization and function of the synaptic AZ within intact neurons in live animals. It will also provide new models for regulation of neurotransmission and short-term synaptic plasticity that integrate the nanoscale dynamics of VGCCs and the structural organization of AZ.